Dataset Information

Identification of Ras-degrading small molecules that inhibit the transformation of colorectal cancer cells independent of ?-catenin signaling.

ABSTRACT: Although the development of drugs that control Ras is an emerging topic in cancer therapy, no clinically applicable drug is currently available. We have previously utilized knowledge of the Wnt/?-catenin signaling-dependent mechanism of Ras protein stability regulation to identify small molecules that inhibit the proliferation and transformation of various colorectal cancer (CRC) cells via degradation of both ?-catenin and Ras. Due to the absence of Ras degradation in cells expressing a nondegradable mutant form of ?-catenin and the need to determine an alternative mechanism of Ras degradation, we designed a cell-based system to screen compounds that degrade Ras independent of the Wnt/?-catenin signaling pathway. A cell-based high-content screening (HCS) system that monitors the levels of EGFP-K-RasG12V was established using HCT-116 cells harboring a nondegradable mutant CTNNB1 (?S45). Through HCS of a chemical library composed of 10,000 compounds and subsequent characterization of hits, we identified several compounds that degrade Ras without affecting the ?-catenin levels. KY7749, one of the most effective compounds, inhibited the proliferation and transformation of CRC cells, especially KRAS-mutant cells that are resistant to the EGFR monoclonal antibody cetuximab. Small molecules that degrade Ras independent of ?-catenin may able to be used in treatments for cancers caused by aberrant EGFR and Ras.

SUBMITTER: Shin W

PROVIDER: S-EPMC5994827 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Identification of Ras-degrading small molecules that inhibit the transformation of colorectal cancer cells independent of β-catenin signaling.

Shin Wookjin W Lee Sang-Kyu SK Hwang Jeong-Ha JH Park Jong-Chan JC Cho Yong-Hee YH Ro Eun Ji EJ Song Yeonhwa Y Seo Haeng Ran HR Choi Kang-Yell KY

Experimental & molecular medicine 20180606 6

Although the development of drugs that control Ras is an emerging topic in cancer therapy, no clinically applicable drug is currently available. We have previously utilized knowledge of the Wnt/β-catenin signaling-dependent mechanism of Ras protein stability regulation to identify small molecules that inhibit the proliferation and transformation of various colorectal cancer (CRC) cells via degradation of both β-catenin and Ras. Due to the absence of Ras degradation in cells expressing a nondegra ...[more]

PMID: 29884842

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Project description:E-cadherin is a tumor suppressor protein with a well-established role in cell-cell adhesion. Adhesion could contribute to tumor suppression either by physically joining cells or by facilitating other juxtacrine signaling events. Alternatively, E-cadherin tumor suppressor activity could result from binding and antagonizing the nuclear signaling function of beta-catenin, a known proto-oncogene. To distinguish between an adhesion- versus a beta-catenin signaling-dependent mechanism, chimeric cadherin constructs were expressed in the SW480 colorectal tumor cell line. Expression of wild-type E-cadherin significantly inhibits the growth of this cell line. Growth inhibitory activity is retained by all constructs that have the beta-catenin binding region of the cytoplasmic domain but not by E-cadherin constructs that exhibit adhesive activity, but lack the beta-catenin binding region. This growth suppression correlates with a reduction in beta-catenin/T cell factor (TCF) reporter gene activity. Importantly, direct inhibition of beta-catenin/TCF signaling inhibits the growth of SW480 cells, and the growth inhibitory activity of E-cadherin is rescued by constitutively activated forms of TCF. Thus, the growth suppressor activity of E-cadherin is adhesion independent and results from an inhibition of the beta-catenin/TCF signaling pathway, suggesting that loss of E-cadherin expression can contribute to upregulation of this pathway in human cancers. E-cadherin-mediated growth suppression was not accompanied by overall depletion of beta-catenin from the cytosol and nucleus. This appears to be due to the existence of a large pool of cytosolic beta-catenin in SW480 cells that is refractory to both cadherin binding and TCF binding. Thus, a small pool of beta-catenin that can bind TCF (i.e., the transcriptionally active pool) can be selectively depleted by E-cadherin expression. The existence of functionally distinct pools of cytosolic beta-catenin suggests that there are mechanisms to regulate beta-catenin signaling in addition to controlling its level of accumulation.

Project description:BACKGROUND:The GTPase KRas4B has been utilized as a principal target in the development of anticancer drugs. PDE6? transports KRas4B to the plasma membrane, where it is released to activate various signaling pathways required for the initiation and maintenance of cancer. Therefore, identifying new small molecules that prevent activation of this GTPase by stabilizing the KRas4B-PDE6? molecular complex is a practical strategy to fight against cancer. METHODS:The crystal structure of the KRas4B-PDE6? heterodimer was employed to locate possible specific binding sites at the protein-protein interface region. Virtual screening of Enamine-database compounds was performed on the located potential binding sites to identify ligands able to simultaneously bind to the KRas4B-PDE6? heterodimer. A molecular dynamics approach was used to estimate the binding free-energy of the complex. Cell viability and apoptosis were measured by flow cytometry. G-LISA was used to measure Ras inactivation. Western blot was used to measure AKT and ERK activation. MIA PaCa-2 cells implanted subcutaneously into nude mice were treated with D14 or C22 and tumor volumes were recorded. RESULTS:According to the binding affinity estimation, D14 and C22 stabilized the protein-protein interaction in the KRas4B-PDE6? complex based on in vitro evaluation of the 38 compounds showing antineoplastic activity against pancreatic MIA PaCa-2 cancer cells. In this work, we further investigated the antineoplastic cellular properties of two of them, termed D14 and C22, which reduced the viability in the human pancreatic cancer cells lines MIA PaCa-2, PanC-1 and BxPC-3, but not in the normal pancreatic cell line hTERT-HPNE. Compounds D14 and C22 induced cellular death via apoptosis. D14 and C22 significantly decreased Ras-GTP activity by 33% in MIA PaCa-2 cells. Moreover, D14 decreased AKT phosphorylation by 70% and ERK phosphorylation by 51%, while compound C22 reduced AKT phosphorylation by 60% and ERK phosphorylation by 36%. In addition, compounds C22 and D14 significantly reduced tumor growth by 88.6 and 65.9%, respectively, in a mouse xenograft model. CONCLUSIONS:We identified two promising compounds, D14 and C22, that might be useful as therapeutic drugs for pancreatic ductal adenocarcinoma treatment.

Dataset Information

Identification of Ras-degrading small molecules that inhibit the transformation of colorectal cancer cells independent of ?-catenin signaling.

Publications

Identification of Ras-degrading small molecules that inhibit the transformation of colorectal cancer cells independent of β-catenin signaling.

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